PROJECT SUMMARY Trichomoniasis is the most prevalent non-viral sexually transmitted disease. Although clinical manifestations of infection are typically mild, the immune system can be concomitantly compromised. Individuals with trichomonal infections have a higher susceptibility to more serious conditions such as cervical cancer, HIV-1, and pelvic inflammatory disease. More recently, trichomonal infection has also been associated with prostate cancer and benign prostatic hyperplasia. The causative agent is the parasitic protozoan Trichomonas vaginalis. Resistance to the existing 5-nitroimidazole class of antitrichomonal drugs has increased markedly in recent years indicating the need for new therapies with novel mechanisms of action. Nucleoside salvage pathway enzymes required by T. vaginalis represent excellent targets for developing novel antitrichomonal agents. The first step in this pathway is the hydrolysis of nucleosides to release the nucleobases. The essential enzymes adenosine/guanosine preferring nucleoside ribohydrolase (AGNH) and uridine nucleoside ribohydrolase (UNH) have been characterized and found to have distinct substrate specificities. Inhibitors of both enzymes were identified using NMR-based activity assays to screen the NIH Clinical Collection. Lack of any significant structural relationship between the two sets of inhibitors further indicates that the two ribohydrolase enzymes are distinct, druggable targets. However, the inhibitors identified from the limited chemistry space sampled by the NIH Clinical Collection have low ligand efficiencies and are thus poor starting points for developing the chemical tools needed for target validation. This project will circumvent this limitation by using a fragment-based approach to define the most critical enzyme/inhibitor interactions. A fragment diversity library encompasses a much broader chemistry space than is contained within the NIH Clinical Collection and will provide the optimal starting points for developing target validation chemical tools. The specific aims of this proposal are: 1) To identify ligand- efficient fragment inhibitors of AGNH and UNH; 2) To identify chemical tools for both AGNH and UNH with IC50 values < 1 ?M; and 3) To validate AGNH and UNH as targets by demonstrating a correlation between enzyme inhibition and antitrichomonal activity. The proposed studies will utilize established NMR methods, computational and medicinal chemistry, and T. vaginalis growth inhibition assays. The overall long-term goals of this project are to validate AGNH and UNH as molecular targets for novel antitrichomonal agents, and to identify inhibitors of these enzymes that possess efficacy against both 5- nitroimidazole-sensitive and 5-nitroimidazole-resistant strains of T. vaginalis.